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Update Date: [2022-09-22]



AHRI report No.17



Department 1.Taiwan Provincial Research Institute for Animai Health1. Taiwan Provincial Research Institute for Animal Health Tamswi, Taiwan, Republic of China.
2. Council for Agricultwal Planning and Development Executive Yuan, Taipei, Taiwan, Republic of China.
Author S. S. Lai1 , C. S. Chen 1, T. H .. Huang1 W.C. Ho 1 and Tracy T. C. Lin 2, 

    Primary swine testicle (ST) eel! was the most susceptible cell to propa-gate the IPC-China strain (IPC) HC vaccine virus. Tl\e virus titer was reached up to 107 ·0 TCID50 /ml and found not increasing significantly throughout 9 passages. Primary swine kidney (SK) and PK-15 cells were also capable of growing the virus, but were less sensitive than primary ST cells. Interestingly, primary rabbit kidney cell and RK-13 cell line were insensitive to grow the rabbit adapted HC virus. The vero cell, a green monkey kidney cell line, also failed to propagate the virus. The homo vin,s interference test (HVIF) and fluorescent antibody cell culture technique-2 step method (FACC:T-2 step) were equal sensitive to detect the virus. 
   Various cell culture system derived from swine tissues have been used for primary isolations and for fundamental studies of HC virus (1,2,3). The tissues mostly used includ-ed kidney, spleen, bone marrow, testicle. ly,:nph-nodes and leucocytes. Replication of the virus was also achieved on bovine kidney cells {4), and chicken fibroblast {5). Both virulent and attenuated HC virus were cul-tivated successfully in cell cultures (6). HC vaccine produced by growing attenuated strain of HC virus was also reported (7 ,8). The LPC strain of HC virus attenuated by serial passage in rabbits for more than 800 passages has been widely used for HC control in, Taiwan (9). However, difficulty in quality control was the disadvantage of vaccine produced from infected tissues. Multiplication of IPC virus in tissue culture of swine origin has been reported (I 0). The LPC virus was non-cytopathic effect (CPE) to the susceptible cells. Therefore 'he HVJF (10, ll) and FACCT-2 step 0.echniques (12) were currently used for de-tecting the IPC ,,irus. The purpose of this study was to seek a suitable cell for growing the LPC virus, which may be used for tissue culture vaccine priduction. 

Viruses IPC-strain HC virus, 816 rabbit passage, was used for the multii:;lication test. Western equine encephalomyelitis (WEE) virus grown in swine testitle cell was used for challenge in the HVIF. GPE" virus, an attenuated HC vaccine virus obtained from the National Institute of Animal Health (NIAH) of Japan, was also grown in ST cell and used in the HVIF.
Cells: Primary ST and SK cells were pre-pared by trypsinization of testes and kidneys obtaL'led from 6-8 weeks old healthy pigs. Primary RK cells was prepared by trypsiniza-tion of kidneys obtained from healthy rabbits. weighted 1-2 kgs. The techniques used for bypsinization . of tissues were previously described (12). PK-15, MVPK of swine qrigin and vero •cell lines were also used for growing the IPC virus.
Multiplication of LPC virus in various types of cells: Confluent monolayers of each type of· cells growing in the 3-liter roller bottles were used fot virus multiplica-lion. Eight ml of !Ox tissue emulsion of the LPC virus infected rabbit spleen containing 106 rabbit infective dose (RID) per ml were used as the initial inoculum for each bottle. After 60 minitues; absorption at 37°C-, the cell sheet was rinsed with PBS and reftlled with 50 ml of Eagle's minimum essential medium (MEM) containing 2% fetal calf serum and irtcubated for S days. After incubation,,the cultured fluid was harvested and the cell shoe! was frozen and thawed for three times. The mixtures of culture fluid and cell debris were used as the inoculum for further passages. Totally six passages were made for each type of cells.
Homo virus interference test (HVIF): The HVIF used for titration of LPC virus was previously described by Lin and Lai (12).
Fluorescent cell culture technique-2 step method (FACCT-2 step): The FACCT-2 step method described previously (12) was used for titration of LPC virus. The virus was grown in the primary ST cells for S days, then the virus fluid was titrated .by trans-ferring to PK-IS cell and stained with HC fluorescent antibody conjugate. Growth curve of LPC virus, ST6 passage: The sixth passage of LPC virus in ST cell (ST6) was used for the growth curve study. The growth curve was determi-ed in ST cells by titrating the mixture of cell associated and cell-free virus using the HVIF and FACCT-2 step techniques. A series of 6 wells plastic plates of ST cell monolayers were infected with LPC virus, ST6 passage at a multiplicity of 10 TCID,,/cell. After 60 minitues' absorption at 37°C, the well were rinsed three times with warm PBS and refilled with 2 ml of MEM containing 2% fetal calf serum. For viral growth curve, at I day in-terval, 6 wells of infected cells and fluid were removed, and frozen and thawed three times. Then the cell debris were removed by centrifugation. The supernatant was used f6r virus titration. 

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